Single-cell sequencing shows cellular heterogeneity of cutaneous lesions in lupus erythematosus

Discoid lupus erythematosus (DLE) and systemic lupus erythematosus (SLE) are both types of lupus, yet the characteristics, and differences between them are not fully understood. Here we show single-cell RNA sequencing data of cutaneous lesions from DLE and SLE patients and skin tissues from healthy controls (HCs). We find significantly higher proportions of T cells, B cells and NK cells in DLE than in SLE. Expanded CCL20+ keratinocyte, CXCL1+ fibroblast, ISGhiCD4/CD8 T cell, ISGhi plasma cell, pDC, and NK subclusters are identified in DLE and SLE compared to HC. In addition, we observe higher cell communication scores between cell types such as fibroblasts and macrophage/dendritic cells in cutaneous lesions of DLE and SLE compared to HC. In summary, we clarify the heterogeneous characteristics in cutaneous lesions between DLE and SLE, and discover some specific cell subtypes and ligand-receptor pairs that indicate possible therapeutic targets of lupus erythematosus.

4. For sub-clustering analysis, using keratinocytes as an example, the authors integrated epidermal cells from two DLE and two SLE patients, plus 3 full-thickness healthy skin from public data and identified 15 sub-clusters in keratinocytes. It is not clear either why they chose to include in their analysis only 3 out of the 5 healthy skin samples from the public dataset. Based on supplementary figure S3, it seems like the samples from HD, DLE, SLE do not mix well, which could be caused by two obvious reasons: (a) the diversity clustering penalty parameter (theta) of the Harmony integration algorithm was not set to a sufficient level to mix healthy keratinocytes cells with keratinocytes in DLE/SLE patients; (b) Harmony is working fine and the sub-clusters do reflect biologically distinct keratinocyte subtypes. Using healthy control samples processed in the same way as the patient's and increasing the number of dermal DLE and SLE samples would be important to bring sound conclusions to these aspects of the study.

5.
Interpreting cell proportions in this study is tricky, as the denominator can vary significantly between conditions (Healthy vs. DLE vs. SLE). This is not a big issue for proportion plots in Fig. 2a or b, because every major cell type is compared against total epidermis/dermis cells. It becomes more complicated in sub-cluster analysis, because for each cell type, its proportion in the total epidermis/dermis can vary drastically. Taking keratinocytes again as an example: according to Fig. 2, proportion for keratinocytes is ~30% and ~75% in epidermis for DLS and SLE, respectively. From Fig.  S1, we can see from the public healthy full-thickness skin, the proportion of keratinocytes ranges from ~25% to ~45%. This means the proportion of keratinocytes in the total skin cells can vary significantly between conditions. Did the authors consider this factor when plotting and interpretating the proportion bar plot for sub-cluster analysis, such as Fig. 3b and other similar plots? As in the comment above, using similarly processed healthy control samples as well as increasing the number of lupus patient samples would enable to get more sound conclusions.
6. Although the authors already did QC and percent.mt filtering and doublet removal at the beginning, it is still worth checking whether in the sub-clustering analysis, any sub-cluster displays high mito percentage, low RNA counts, as well as evaluate the possibility of leftover doublet clusters by checking known markers on all sub-clusters.  Fig. 6I seems to be a repeat of 6D rather than the display of dermal B cell subcluster data. Figures 9 A and D are unreadable. Also, supplement table S1 was split across several pages, which is difficult for readers to follow.
Reviewer #3 (Remarks to the Author): Zheng et al show for the first time the characterization of the cell types present in the skin of patients with DLE and SLE, at the single cell RNA sequencing level. This adds valuable information to our knowledge about both which cell types are present, and the differences between the two manifestations of lupus lesions. That being said, I have some major and minor reservations about this article.
Major points: 1. General interest of reading style. Considering the paper is largely built of same technique used 6 times for 6 different cells types (keratinoyctes, fibroblasts, T cells, B cells, DCs and NK cells), the manuscript reads slightly monotonously in my opinion. Although I can appreciate how thorough this is, it is difficult to hold attention. The flow of the article is also sometimes jumpy, for example sudden mention of the potential involvement of anti-virus response in LE lesions (line 216), which seems to come from nowhere. 2. N numbers -only 2 patients in epidermal group for both DLE and SLE were analyzed. I really doubt if you make the conclusions in this article based on this number of patients? Table S1 with the clinical information is also not readable at all. 3. Also use of 'SC' for clusters of all cell types analyzed, no matter whether keratinocyte, fibroblast, T cell, B cell, DC or NK cells makes reading difficult? Not sure how to remedy this, you need to call them something, and there are lots of them? 4. Over interpretation, for example: Conclusion strong? Line 182 -found to be involved in local infiltration of immune cells -this has not been demonstrated by the authors. Only that they expressed genes suggesting this. Also -infiltration is not physically close to these cells, or not? 5. Issues with Figure 9.  Table S1. 5. SC7 proliferating keratinocyte sub group -did the authors also look at expression of classical markers of proliferation in this cluster, for example Ki67 or PCNA? 6. Pseudotime trajectory Fig. 3g -very minimal information -can the authors put more labels in at least? 7. Are these skin lesions with scar formation? Would be interesting to know, because of this link with activated types of fibroblasts. 8. Also how many tissues were stained for these validation experiments in Figures 2-4, and why was this not performed in the other cell subsets (T cells, B cells, DCs, NK cells)? 1 We deeply appreciate you and the reviewers for their encouraging and insightful 2 suggestions and comments on improving our manuscript. As requested, we have 3 performed additional experiments as described in detail below and added the 4 explanation for some questions. All the changes have been highlighted in red font in 5 the revised manuscript. With these extensive revisions, we believe that our manuscript 6 has been significantly improved. 7 8 9 Reviewer 1: 10 This is a very good manuscript that reveals several interesting features of cells in the 11 dermis and epidermis in both SLE and discoid LE. Both diseases have cutaneous lesions, 12 but they are known to differ, however no study had looked before at the comparison. 13 This paper is good, quite well written and the descriptions are clear. As every single 14 cell work, it is very descriptive. 15 Some interesting findings are cells other that pDCs producing IFN, expansion of 16 fibroblasts, infiltration of neutrophils, as well as infiltrating T cells also with an IFN 17 response (ISG+GPR183+HSPhi). Also, the pseudotime analysis is interesting and 18 shows the trajectories of the various cells in the tissue. 19 20 1. when describing the several pathways found using GO, please do not write "and so 21 on". This is not serious. If you do not want to describe all the pathways found, show a 22 This is an interesting study that brings provocative data that could potentially shed light 69 on the pathogenesis of two different types of lupus skin involvement. The authors 70 analyzed scRNA-Seq data from epidermis and dermis cells in SLE and DLE patients 71 and compared the data with a public dataset of full thickness skin biopsies from 3 72 healthy donors. They also use limited immunohistochemistry to validate some of their 73 transcriptional results. The authors first analyzed major skin cell clusters from total 74 epidermis or dermis, followed by breakdown and sub-cluster analysis of each cell type. 75 The overall methodology for scRNA-Seq data processing and integration to detect 76 major skin cell types looks sound, yet details about sub-cluster analysis are insufficient. there are validated measures of lupus skin disease activity (mCLASI) that would be very 82 important to incorporate into the analysis. Information on the location of the 83 rash/biopsies would also be desirable. This is particularly important as it has been shown that skin regions exposed to UV light display a different transcriptional profile 85 compared to the regions that are not (doi: 10.1038/s42003-020-0922-4). Overall, the 86 display of the currently included clinical and laboratory values should be improved. proportion plots in Fig. 2a or b, because every major cell type is compared against total epidermis/dermis cells. It becomes more complicated in sub-cluster analysis, 141 because for each cell type, its proportion in the total epidermis/dermis can vary 142 drastically. Taking keratinocytes again as an example: according to Fig. 2, proportion  143 for keratinocytes is ~30% and ~75% in epidermis for DLE and SLE, respectively. From 144 shown in the revised Fig.3b, Fig.4b, Fig.5b, Fig.5g, Fig.6b, Fig.6g, Fig.7b, Fig.7g, 160  Response: We thank for the suggestion of the reviewer. As suggested, we check the 170 quality of data for the sub-clustering analysis and did not find the high mito percentage 171 and low RNA counts in the sub-clustering analysis. Here, we take the epidermal T cell 172 sub-clustering results as an example to show the features (200-5000), mito percentage 173 (<20%) and the percent of red cells (<10%) in epidermal T cells performed sub-174 clustering analysis (as shown below). In addition, we checked the known markers 175 expression on all sub-clusters. We did not find the leftover doublet cluster in most of 176 sub-clusters. However, we found that the tiny proportions of T cells, B cells and 177 Macro/DC subclusters in epidermis expressed the keratinocyte marker KRT1 or KRT14 178 ( Fig S3a, Fig S4a, S4e, Fig S5a, S5f, Fig S6a, S6e, Fig S7a,  Although I can appreciate how thorough this is, it is difficult to hold attention. The flow 207 of the article is also sometimes jumpy, for example sudden mention of the potential 208 involvement of anti-virus response in LE lesions (line 216), which seems to come from 209 nowhere. 210 211 Response: We deeply appreciate the reviewer's insightful comment and suggestion. In 212 the manuscript, we displayed the cell atlas of epidermis and dermis in skin lesions of 213 DLE and SLE patients. We showed the cell subclusters, gene lists, and GO pathways of 214 6 different cell types, which will help us understand the pathogenesis of lupus cutaneous 215 lesion and the difference in cutaneous lesion between DLE and SLE. To avoid the 216 monotonous description in the results, we try our best to improve the writing style to 217 make it more attractive. And we also revised these sentences to make their presence 218 more reasonable. 219 220 2. N numbers -only 2 patients in epidermal group for both DLE and SLE were analyzed. 221 I really doubt if you make the conclusions in this article based on this number of patients? 222 Table S1 with the clinical information is also not readable at all. 223 224 Response: We agree with the reviewer. In order to enhance the reliability of the 225 conclusions, we added the epidermis samples from 3 DLE patients and 3 SLE patients. 226 The new results of comparison in epidermis were obtained from 5 DLE patients, 5 SLE 227 patients and 4 healthy subjects in the revised manuscript. We are sorry for the 228 unreadable table S1. We have revised the Table S1  about interactions, we replaced the Fig. 9a, b, e and f with bubble plots (Fig. 9c, 9d) 259 which showed different mean expression levels of several ligand-receptor pairs in DLE, 260 SLE and HC. The Fig. 9c, d, g, and h were improved to present the difference in strength 261 of cell interactions among DLE, SLE and HC ( Fig. 9a and 9b). In addition, we also 262 performed the immune-staining of ligand and receptor in the skin tissues of DLE, SLE 263 and HC to validate our findings in cell interactions analysis (Fig. 9e)  Missing from Table S1. 300 301 Response: In the revised manuscript, we collected the skin samples of healthy controls 302 for scRNA-seq according to the reviewer's suggestion. The ages of healthy controls 303 were added in the revised Table S1. 304 305 5. SC7 proliferating keratinocyte sub groupdid the authors also look at expression 306 of classical markers of proliferation in this cluster, for example Ki67 or PCNA? 307 308 Response: We thank the reviewer's suggestion. We found that the classical proliferation 309 markers such as MKI67(Ki67) and PCNA were highly expressed in the cycling 310 keratinocytes (Fig. 3c). 311 312 6. Pseudotime trajectory Fig. 3g very minimal informationcan the authors put more labels in at least? 314 315 Response: We agree with the reviewer. We added the labels in pseudotime trajectory of 316 revised Fig. 3g. 317 318 7. Are these skin lesions with scar formation? Would be interesting to know, because of 319 this link with activated types of fibroblasts. 320 321 Response: We thank for the comment. These skin lesions from patients did not contain 322 scar formation, which was shown in Table S1.